Many endogenous factors affect peripheral sensory innervation. Deficiency in one endogenous factor, vitamin D, is linked clinically to musculoskeletal pain, but the mechanisms are poorly understood. The working hypothesis of this project is that a vitamin D deficiency leads to nociceptor hyperinnervation of tissue, altering pain threshold. Understanding whether vitamin D directly affects peripheral neurons will enable physicians to orchestrate more effective pain prevention and treatment strategies and provide further insight into the basic science of peripheral sensory plasticity. The role of vitamin D in nociceptor modulation will be addressed using a rat model in three specific aims: Aim 1) Identify which DRG subpopulations express machinery for vitamin D signaling and metabolism. Immunofluorescence and real time RT PCR of laser- captured retrograde-labeled adult DRG neurons will be used to calculate the percentage of marker and projection target-specific subpopulations that express vitamin D receptor (VDR) and 1 alpha-hydroxylase. The prevalence for widespread pain is especially high in hypoestrogenaemic women, suggesting a hormonal component. Therefore the expression levels of these proteins will be examined in staged cycling and ovariectomy induced menopause rats. Aim 2) Determine whether vitamin D deficiency induces hyperinnervation of muscle and increased musculoskeletal pain before signs of bone pathology in the rat. Using vitamin D deficient diets in combination with pain behavior analyses, the time course of hypovitaminosis D induced pain-related behavior will be mapped. Changes in innervation density will be examined through morphometric analysis of immunoreactive axonal outgrowth in tissue. Spinal dorsal horn c-fos expression will be used to determine if there is increased peripheral input to the CNS. Bone calcification will be analyzed at the onset of pain-related behavior in order to determine whether nociception is initiated in the absence of bone pathology. In addition, using diets with modified calcium and phosphate content, the role of altered serum electrolyte levels in hypovitaminosis D-induced pain can be deciphered. Aim 3) Evaluate the effects of physiological and patho-physiological levels of vitamin D on neurite outgrowth in primary DRG culture. Neurite outgrowth in adult dissociated DRG cultures will be determined by morphometric analysis of immunoreactive neurites. In addition, neurite chamber cultures (Campenot cultures) will be used to determine whether vitamin D initiates its effects within the soma or the neurite. PUBLIC HEALTH RELEVANCE: Vitamin D deficiency is associated with muscle pain;understanding the mechanisms underlying this phenomenon may provide insight into treatment strategies and advance the science of sensory function.